Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp- mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp- mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

Bio21 Molecular Science and Biotechnology Institute School of BioSciences University of Melbourne Melbourne 3010 Australia

Biology Centre Czech Academy of Sciences Institute of Entomology Branišovská 31 370 05 České Budějovice Czech Republic

Canadian Centre for Computational Genomics McGill University Montreal QC Canada

Centre for Genomic Research Institute of Integrative Biology The Biosciences Building Crown Street L69 7ZB Liverpool United Kingdom

Department of Environmental Engineering University of Patras 2 Seferi str 30100 Agrinio Greece

Department of Insect Biotechnology in Plant Protection Justus Liebig University Gießen Institute for Insect Biotechnology Winchesterstr 2 35394 Gießen Germany

Insect Pest Control Laboratory Joint FAO IAEA Programme of Nuclear Techniques in Food and Agriculture Seibersdorf 1400 Vienna Austria

McGill University Genome Centre McGill University Montreal QC Canada

School of Biological Sciences University of Adelaide 5005 Adelaide Australia

South Australian Research and Development Institute Waite Road Urrbrae 5064 South Australia

USDA ARS Daniel K Inouye US Pacific Basin Agricultural Research Center 64 Nowelo Street Hilo HI 96720 USA

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